Plant Biotechnology Vol.32 No.4

Contents

Original Papers

Pleiotropic phenotype of transgenic Arabidopsis plants that produce the LOV domain of LOV KELCH PROTEIN2 (LKP2)

Tomoyuki Takase, Yuji Miyazaki, Masahiro Yasuhara, Shunya Mitsui, Tomohiro Kiyosue. . . . . . . 273 [PDF]

LOV KELCH PROTEIN2 (LKP2) is a blue-light receptor protein composed of three functional domains: a light, oxygen, or voltage (LOV) domain, an F-box motif (F), and Kelch repeats. LKP2 is postulated to be a component of an SCF complex and function in ubiquitination of proteins that control the circadian clock and photoperiodic flowering. Transgenic Arabidopsis plants that produce LOV, F, or a combination of LOV and F fused to green fluorescent protein (named GL, GF, and GLF, respectively) were produced using constructs containing the Cauliflower mosaic virus 35S promoter. Under continuous white light, the circadian rhythms of control and GF plants were similar, whereas those of GL and GLF plants were shorter. Under continuous red light, the hypocotyl lengths of control and GF seedlings were similar, whereas that of GL seedlings was longer. Late flowering and down-regulation of CONSTANS and FLOWERING LOCUS T were observed in GL and GLF plants compared to GF and control plants under long-day conditions. These results suggest that the previously reported pleiotropic phenotype of LKP2-overproducing plants, which show altered circadian rhythm, hypocotyl elongation, and photoperiodic flowering, is not only due to the promotion of ubiquitination and subsequent degradation of substrate proteins of the SCFLKP2 complex but may also be due to the functional disruption of regulatory proteins that interact with LKP2 LOV.

Overexpression of the phosphoenolpyruvate carboxykinase gene (SlPEPCK) promotes soluble sugar accumulation in fruit and post-germination growth of tomato (Solanum lycopersicum L.)

Yong-Xing Huang, Yukihisa Goto, Satoko Nonaka, Naoya Fukuda, Hiroshi Ezura, Chiaki Matsukura. . . . . . . 281 [PDF]

Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme that regulates the gluconeogenesis pathway in plants. While the biochemical properties of PEPCK have been reported for many species, its physiological function is not fully understood in plants with fresh berry-type fruit. To clarify its physiological role(s) in the tomato plant, the effect of excessive PEPCK was investigated using transgenic lines overexpressing SlPEPCK by either the CaMV 35S constitutive promoter or the fruit-specific E8 promoter. Detailed characterization of the phenotypic and metabolic properties of the 35S promoter-driven lines revealed that the transgenic seedlings exhibited earlier germination and better seedling growth compared with the wild type. Interestingly, seedling growth at 10 days after sowing of the transgenic lines was enhanced by an exogenous sucrose supply. These results suggest that PEPCK enhances seedling growth through PEPCK/pyruvate kinase-mediated pathway rather than gluconeogenesis during germination. In addition, increased soluble sugars and decreased malate contents were observed in red-ripe fruit in both the 35S and E8 promoter-driven lines, indicating the participation of gluconeogenesis in sugar/acid metabolism during fruit ripening. The present results are totally opposite to those observed in PEPCK-suppressed RNAi lines, which were investigated in our previous work. The results indicate the regulatory role of PEPCK in post-germination growth and sugar/organic acid accumulation in ripening tomato fruit.

Co-expression of microbial cellulases in transgenic wheat as a potential source for cellulosic ethanol production

Hashmath I. Hussain, Gregory D. Nugent, Kim Stevenson, David M. Stalker, Trevor W. Stevenson. . . . . . . 291 [PDF]

Crop residues are produced in abundance in agriculture and forestry and are a potential source of lignocellulosic biomass for ethanol production. Recent research has included the heterologous expression in transgenic plants of genes encoding cellulases to produce fermentable sugars from plant biomass. In this study, transgenic wheat lines were generated co-expressing the genes for an endoglucanase 1 (E1) of Acidothermus cellulolyticus and cellobiohydrolase 1 (CBH1) of Trichoderma reesei. Both genes were under the control of a wheat Rubisco small subunit promoter (RbcS). Transgenic wheat leaves accumulated apoplast targeted E1 and CBH1 proteins at levels up to 1 and 0.5% respectively of total soluble protein as determined by immunoblotting. Transgenic plants co-expressing E1 and CBH1 were analysed by a 4-methylumbelliferyl-β-D-cellobioside (MUC) assay and enzymatic activity was detected up to 92 nmol 4-MU/mg tsp/min.

Identification of furostanol glycoside 26-O-β-glucosidase involved in steroidal saponin biosynthesis from Dioscorea esculenta

Masaru Nakayasu, Takashi Kawasaki, Hyoung Jae Lee, Yukihiro Sugimoto, Michio Onjyo, Toshiya Muranaka, Masaharu Mizutani. . . . . . . 299 [PDF]

Steroidal saponins are natural surfactants with various biological activities, and the tubers of Dioscorea, known as yam, contain a furostanol glycoside protodioscin and a spirostanol glycoside dioscin, which are valuable saponins required for semi-synthetic production of pharmaceutical steroidal drugs. Steroidal saponins are biosynthesized from cholesterol via several steps of oxygenation and transglycosylation, and a β-glucosidase is involved in the hydrolytic conversion from furostanol glycosides to spirostanol glycosides. To investigate steroidal saponin biosynthesis in Dioscorea spps, comparative transcriptome analysis of high saponin producers, D. esculenta and D. cayenensis, and a low producer, D. alata, was performed using 454 pyrosequencing. In this study, we isolated and characterized a β-glucosidase (DeF26G1) from D. esculenta. The DeF26G1 cDNA encodes a family 1 glucosidase, and the DeF26G1 transcript was present at high levels in D. esculenta but not detected in D. alata. The recombinant DeF26G1 protein hydrolyzed the 26-O-glycosidic bond of protodioscin to form dioscin, indicating that the DeF26G1 gene encodes furostanol glycoside 26-O-β-glucosidase. These results suggested that DeF26G1 is involved in the conversion of furostanol saponins to spirostanol saponins, which seems to be related to biological defense response in the leaves of Dioscorea plants.

Compatibility of translational enhancers with various plant species

Takeshi Matsui, Kazutoshi Sawada, Eiji Takita, Ko Kato. . . . . . . 309 [PDF]

Translational enhancers are effective tools to increase the expression level of transgenes in plant cells, and some candidate elements are in use today. However, knowledge about suitable elements for a given plant species are limited. We aim here to make a catalogue of translational enhancers, and evaluated the effectiveness of 5′-UTRs of Nicotiana tabacum alcohol dehysrogenase gene (NtADH 5′-UTR), Arabidopsis thaliana ADH gene (AtADH 5′-UTR), Oryza sativa ADH gene (OsADH 5′-UTR), Ω derived from tobacco mosaic virus, and tobacco etch virus leader (TEVL) in various plant species. We found that the OsADH 5′-UTR is functional in all plant species tested, and that Ω and TEVL are effective in eudicots but not in monocots. From these results, we speculate that the degree of translational enhancement of any element in a given plant species is closely correlated with the phylogenic position of the species.

Short Communication

Effect of the 5′-untranslated region on the expression of transgene in liverwort, Marchantia polymorpha

Miho Takemura, Shingo Nagaya, Haruka Kida. . . . . . . 317 [PDF]

Transgenic plants have the potential to provide substantial benefits in the production of pharmaceutical compounds, valuable nutrition, and vaccines. Cauliflower mosaic virus 35S promoter is the most general promoter in higher plants, and also used to express the transgene in liverwort. However, some genes, such as those found in other species, are difficult to express in liverwort. We reported previously that the 5′-untranslated region (UTR) of liverwort ADH-like glucose dehydrogenase gene enhanced transient gene expression in liverwort. In this study, we tested the effects of 5′-UTR on the expression of Gracilaria vermiculophylla cyclooxygenase (GvCOX) gene in stably transformed liverwort. The GvCOX gene encodes the enzyme to produce prostaglandins used as pharmaceutical compounds. Cyclooxygenase activity in transgenic liverwort without 5′-UTR was low, although this mRNA was accumulated. In contrast, addition of the 5′-UTR to the GvCOX gene significantly increased cyclooxygenase activity in transgenic liverwort. We also found that the boundary sequences between the 5′-UTR and GvCOX gene affected mRNA accumulation level. Our system with the 5′-UTR provides a powerful tool for enhancing transgene expression in liverwort.

Notes

High efficiency Agrobacterium-mediated transformation of Dendrobium orchid using protocorms as a target material

Wasana Phlaetita, Dong Poh Chin, Ntui Valentine Otang, Ikuo Nakamura, Masahiro Mii. . . . . . . 323 [PDF]

Dendrobium orchid is one of the most popular cut flower and potted plants. In this study, a protocol for efficient genetic transformation of D. nobile-type orchids was established by co-cultivating 21 day-old protocorms for 3 days with Agrobacterium tumefaciens strain EHA101 carrying pIG121Hm harboring β-glucuronidase (GUS) gene as reporter gene and hygromycin phosphotransferase (hpt) gene as selectable marker gene. After selection of the infected protocorms on New Dogashima (ND) medium containing 10 g l−1 maltose, 30 mg l−1 hygromycin and 20 mg l−1 meropenem for 3 months followed by the culture on hygromycin-free recovery medium for 1 month, secondary protocorm-like bodies (PLBs) produced on this medium were again transferred onto secondary selection (regeneration) medium. Plantlets were successfully regenerated from these secondary PLBs after the transfer. The highest transformation efficiency of 27.3% was obtained when protocomrs were inoculated with 10 times diluted Agrobacterium solution (OD600=0.1) for 300 min. Transformation of the selected plants was confirmed by GUS assay, PCR and Southern blot analysis. This protocol could be adopted to produce transgenic D. nobile-type orchids with various traits such as novel flower color and resistances to biotic and abiotic stresses.

Efficacy of transgenic rice containing human interleukin-10 in experimental mouse models of colitis and pollen allergy

Hidenori Takagi, Nobumasa Watanabe, Takachika Hiroi, Fumio Takaiwa. . . . . . . 329 [PDF]

Recombinant human interleukin-10 (hIL-10) is highly expressed in transgenic rice endosperm and forms active homodimers in the ER-derived hIL-10 body. In this study, we examined the preclinical efficacy of transgenic rice accumulating hIL-10 (hIL-10 rice) in in vivo experimental mouse models of colitis and pollen allergy. In the group of mice orally fed hIL-10 rice, the development of Japanese cedar pollen allergen-specific IgE and splenic T cell responses were significantly inhibited. In addition, oral feeding of hIL-10 rice showed therapeutic as well as prophylactic efficacy against experimental colitis developed in IL-10-deficient mice. These results indicate the clinical potentiality of hIL-10 rice for the control of inflammatory and allergic disorders through efficient delivery of hIL-10 to the gut-associated lymphoid tissue.

A highly efficient AgarTrap method for genetic transformation of mature thalli of the liverwort Marchantia polymorpha L.

Shoko Tsuboyama-Tanaka, Satoko Nonaka, Yutaka Kodama. . . . . . . 333 [PDF]

Marchantia polymorpha L. has been established as a model liverwort species for understanding plant evolution, and several genetic transformation technologies have been developed. Recently, we described a simplified Agrobacterium-mediated genetic transformation method termed AgarTrap (Agar-utilized Transformation with Pouring Solutions) that uses sporelings (S-AgarTrap) or gemmae/gemmalings (G-AgarTrap), both of which easily produce a sufficient number of independent transformants. In this study, we report the development of a novel AgarTrap protocol using mature pieces of liverwort thalli (T-AgarTrap). Optimal transformation efficiency was approximately 70%, and 100% transformation was achieved in a few experimental cases. The efficiency of T-AgarTrap was much greater than those of S- and G-AgarTrap methods. This highly efficient T-AgarTrap protocol seems to promote molecular research with M. polymorpha.

Physical interaction between SnRK2 and PP2C is conserved in Populus trichocarpa

Xueqing Song, Misato Ohtani, Chiaki Hori, Arika Takebayasi, Ryoko Hiroyama, Nur Ardiyana Rejab, Takaomi Suzuki, Taku Demura, Tongming Yin, Xiang Yu, Qiang Zhuge. . . . . . . 337 [PDF]

Type 2C protein phosphatase (PP2C) is a central player in abscisic acid (ABA) signaling transduction, which is required for plant growth, development and stress responses. In Arabidopsis, group A PP2Cs inhibit activity of the SNF1-related protein kinases 2 (SnRK2) family via physical interaction. To clarify whether this scheme is conserved in woody plants, we experimentally isolated the genes homologous to three members of group A PP2Cs (PtABI1, PtAHG1 and PtAHG3) and 12 SnRK2s (PtSnRK2.1–2.12) from a model tree Populus trichocarpa, and examined their interaction using a yeast two-hybrid assay. Our results showed that only three PtSnRK2 proteins had a positive interaction with PtPP2Cs: PtSnRK2.10 possessed strong interaction activity with all three PtPP2Cs, while significant, but relatively weak, interactions were observed with PtSnRK2.6 and PtSnRK2.9. These three PtSnRK proteins are grouped into subclass 2 or 3, which are considered to be ABA-dependent kinases in Arabidopsis. These findings suggest that physical interaction between SnRK2 and PP2C is also conserved in poplars and may be involved in the ABA signaling pathway in tree plants.

Possible contribution of TED6 and TED7, secondary cell wall-related membrane proteins, to evolution of tracheary element in angiosperm lineage

Nur Ardiyana Rejab, Yoshimi Nakano, Arata Yoneda, Misato Ohtani, Taku Demura. . . . . . . 343 [PDF]

The Tracheary Element Differentiation-Related6 (TED6) and TED7 membrane proteins function in the differentiation of xylem vessel elements, the cellular units for water conduction in angiosperm plants. Functional analysis of TED6 and TED7 had suggested that these proteins directly bind to a subunit of the secondary cell wall (SCW)-related cellulose synthase complex, to promote SCW formation in xylem vessel elements. However, whether TED6 and TED7 function in SCW formation of xylem vessel elements only, or function broadly in other cell types has remained unclear. To clarify this, we conducted detailed expression analysis of TED6 and TED7 genes in Arabidopsis thaliana. This showed that TED6 and TED7 are expressed in differentiating vessel elements of all organs examined here, including roots, leaves, and inflorescence stems. We detected no TED6 and TED7 promoter activity in other types of cells with SCW thickening, such as fiber cells and anther endothecium, indicating that TED6 and TED7 have specific roles in SCW formation of vessel elements. Homology searches identified TED6/7-like proteins only in the angiosperm lineage. These data suggest that development of TED6/7 proteins could have coincided with the emergence of the angiosperm lineage, and that TED6/7 may have made key contributions to the evolution of water-conducting cells from tracheids to vessels.

Generation of transgenic Linum perenne by Agrobacterium-mediated transformation

Kohji Murase, Yuki Sugai, Shinya Hayashi, Yasunao Suzuki, Koichi Tsujii, Seiji Takayama. . . . . . . 349 [PDF]

Here we report new methods for tissue culture, plant regeneration, and Agrobacterium-mediated transformation of perennial flax (Linum perenne). To generate transgenic plants, L. perenne hypocotyls were co-cultivated with Agrobacterium harboring a binary vector (pBI121 or pRI909) for 2 days. The transgenic calli and shoots were induced on Murashige–Skoog medium supplemented with 10 µM 6-benzylaminopurine and 0.3 µM 1-naphthalenacetic acid under selection with 100 mg l−1 kanamycin. For root induction from the shoots, 0.1 mg l−1 indole-3-butyric acid was found to be most efficient. We transformed four types of binary vectors encoding a florigen gene under the control of 35S or phloem-specific promoters. Transgenic plants were obtained with high efficiency (4–24%) and exhibited early flowering phenotypes. Our transformation method will contribute to future studies of L. perenne that require transgenic plants.